Causative agent of the Mystery Swine Disease, vaccine compositions and diagnostic kits

ABSTRACT

Composition of matter comprising the causative agent of Mystery Swine Disease, Lelystad Agent, in a live, attenuated, dead, or recombinant form, or a part or component of it. Vaccine compositions and diagnostic kits based thereon. Recombinant nucleic acid comprising a Lelystad Agent-specific nucleotide sequence. Peptides comprising a Lelystad Agent-specific amino acid sequence. Lelystad Agent-specific antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 10/226,065, now U.S. Pat. No. 6,806,086, filed Aug.21, 2002, which is a divisional of U.S. application Ser. No. 09/565,864,filed May 5, 2000, now U.S. Pat. No. 6,455,245, issued Sep. 24, 2002,which is a divisional of U.S. application Ser. No. 08/747,863, filedNov. 13, 1996, now U.S. Pat. No. 6,197,310, issued Mar. 6, 2001, whichitself is a divisional of U.S. patent application Ser. No. 08/157,005,filed Nov. 26, 1993, now U.S. Pat. No. 5,620,691, which is a U.S.National Stage under 35 U.S.C. § 371 of International Patent ApplicationPCT/NL92/00096, filed Jun. 5, 1992, the contents of all of which areincorporated by this reference.

TECHNICAL FIELD Field of the Invention

The invention relates to the isolation, characterization and utilizationof the causative agent of the Mystery Swine Disease (MSD). The inventionutilizes the discovery of the agent causing the disease and thedetermination of its genome organization, the genomic nucleotidesequence and the proteins encoded by the genome, for providingprotection against and diagnosis of infections, in particular,protection against and diagnosis of MSD infections, and for providingvaccine compositions and diagnostic kits, either for use with MSD orwith other pathogen-caused diseases.

BACKGROUND

In the winter and early spring of 1991, the Dutch pig industry wasstruck by a sudden outbreak of a new disease among breeding sows. Mostsows showed anorexia, some aborted late in gestation (around day 110),showed stillbirths or gave birth to mummified fetuses and some hadfever. Occasionally, sows with bluish ears were found, therefore, thedisease was commonly named “Abortus Blauw”. The disease in the sows wasoften accompanied by respiratory distress and death of their youngpiglets and often by respiratory disease and growth retardation of olderpiglets and fattening pigs.

The cause of this epizootic was not known, but the symptoms resembledthose of a similar disease occurring in Germany since late 1990, andresembled those of the so-called “Mystery Swine Disease” as seen since1987 in the mid-west of the United States of America and in Canada(Hill, 1990). Various other names have been used for the disease; inGermany it is known as “Seuchenhafter Spätabort der Schweine” and inNorth America it is also known as “Mystery Pig Disease”, “MysteriousReproductive Syndrome”, and “Swine Infertility and RespiratorySyndrome”. In North America, Loula (1990) described the general clinicalsigns as:

-   -   1) off feed, sick animals of all ages;    -   2) abortions, stillbirths, weak pigs, mummies;    -   3) post-farrowing respiratory problems; and    -   4) breeding problems.

No causative agent has as yet been identified, but encephalomyocarditisvirus (“EMCV”), porcine parvo virus (“PPV”), pseudorabies virus (“PRV”),swine influenza virus (“SIV”), bovine viral diarrhea virus (“BVDV”), hogcholera virus (“HCV”), porcine entero viruses (“PEV”), an influenza-likevirus, chlamidiae, leptospirae, have all been named as a possible cause(Loula, 1990; Mengeling and Lager, 1990; among others).

SUMMARY OF THE INVENTION

The invention provides a composition of matter comprising isolatedLelystad Agent which is the causative agent of Mystery Swine Disease,the Lelystad Agent essentially corresponding to the isolate LelystadAgent (CDI-NL-2.91) deposited Jun. 5, 1991 with the Institut Pasteur,Collection Nationale de Cultures De Microorganismes (C.N.C.M.) 25, ruedu Docteur Roux, 75724—Paris Cedex 15, France, deposit number I-1102.The words “essentially corresponding” refer to variations that occur innature and to artificial variations of Lelystad Agent, particularlythose which still allow detection by techniques like hybridization, PCRand ELISA, using Lelystad Agent-specific materials, such as LelystadAgent-specific DNA or antibodies.

The composition of matter may comprise live, killed, or attenuatedisolated Lelystad Agent; a recombinant vector derived from LelystadAgent; an isolated part or component of Lelystad Agent; isolated orsynthetic protein (poly)peptide, or nucleic acid derived from LelystadAgent; recombinant nucleic acid which comprises a nucleotide sequencederived from the genome of Lelystad Agent; a (poly)peptide having anamino acid sequence derived from a protein of Lelystad Agent, the(poly)peptide being produced by a cell capable of producing it due togenetic engineering with appropriate recombinant DNA; an isolated orsynthetic antibody which specifically recognizes a part or component ofLelystad Agent; or a recombinant vector which contains nucleic acidcomprising a nucleotide sequence coding for a protein or antigenicpeptide derived from Lelystad Agent.

On the DNA level, the invention specifically provides a recombinantnucleic acid, more specifically recombinant DNA, which comprises aLelystad Agent-specific nucleotide sequence shown in FIG. 1 (SEQ IDNO:1) which includes FIGS. 1 a through 1 q. Preferably, the LelystadAgent-specific nucleotide sequence is selected from any one of the ORFs(Open Reading Frames) shown in FIG. 1 (SEQ ID NO:1).

On the peptide/protein level, the invention specifically provides apeptide comprising a Lelystad Agent-specific amino acid sequence shownin FIG. 1 (SEQ ID NO:1).

The invention further provides a vaccine composition for vaccinatinganimals, in particular mammals, more in particular pigs or swine, toprotect them against Mystery Swine Disease, comprising Lelystad Agent,either live, killed, or attenuated; or a recombinant vector whichcontains nucleic acid comprising a nucleotide sequence coding for aprotein or antigenic peptide derived from Lelystad Agent; an antigenicpart or component of Lelystad Agent; a protein or antigenic polypeptidederived from, or a peptide mimicking an antigenic component of, LelystadAgent; and a suitable carrier or adjuvant.

The invention also provides a vaccine composition for vaccinatinganimals, in particular mammals, more in particular pigs or swine, toprotect them against a disease caused by a pathogen, comprising arecombinant vector derived from Lelystad Agent, the nucleic acid of therecombinant vector comprising a nucleotide sequence coding for a proteinor antigenic peptide derived from the pathogen, and a suitable carrieror adjuvant.

The invention further provides a diagnostic kit for detecting nucleicacid from Lelystad Agent in a sample, in particular a biological samplesuch as blood or blood serum, sputum, saliva, or tissue, derived from ananimal, in particular a mammal, more in particular a pig or swine,comprising a nucleic acid probe or primer which comprises a nucleotidesequence derived from the genome of Lelystad Agent, and suitabledetection means of a nucleic acid detection assay.

The invention also provides a diagnostic kit for detecting antigen fromLelystad Agent in a sample, in particular a biological sample such asblood or blood serum, sputum, saliva, or tissue, derived from an animal,in particular a mammal, more in particular a pig or swine, comprising anantibody which specifically recognizes a part or component of LelystadAgent, and suitable detection means of an antigen detection assay.

The invention also provides a diagnostic kit for detecting an antibodywhich specifically recognizes Lelystad Agent in a sample, in particulara biological sample such as blood or blood serum, sputum, saliva, ortissue, derived from an animal, in particular a mammal, more inparticular a pig or swine, comprising Lelystad Agent; an antigenic partor component of Lelystad Agent; a protein or antigenic polypeptidederived from Lelystad Agent; or a peptide mimicking an antigeniccomponent of Lelystad Agent; and suitable detection means of an antibodydetection assay.

The invention also relates to a process for diagnosing whether ananimal, in particular a mammal, more in particular a pig or swine, iscontaminated with the causative agent of Mystery Swine Disease,comprising preparing a sample, in particular a biological sample such asblood or blood serum, sputum, saliva, or tissue, derived from theanimal, and examining whether it contains Lelystad Agent nucleic acid,Lelystad Agent antigen, or antibody specifically recognizing LelystadAgent, the Lelystad Agent being the causative agent of Mystery SwineDisease and essentially corresponding to the isolate Lelystad Agent(CDI-NL-2.91) deposited Jun. 5, 1991 with the Institut Pasteur, Paris,France, deposit number I-1102.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a result of combined efforts of the Central VeterinaryInstitute (CVI) and the Regional Animal Health Services (RAHS) in theNetherlands in trying to find the cause of the new disease MSD. Farmswith pigs affected by the new disease were visited by fieldveterinarians of the RAHS. Sick pigs, specimens of sick pigs, and sowsera taken at the time of the acute and convalescent phase of thedisease were sent for virus isolation to the RAHS and the CVI. Pairedsera of affected sows were tested for antibodies against ten knownpig-viruses. Three different viruses, encephalomyocarditis virus,porcine entero virus type 2, porcine entero virus type 7, and an unknownagent, Lelystad Agent (LA), were isolated. Sows which had reportedlybeen struck with the disease mainly seroconverted to LA, and rarely toany of the other virus isolates or the known viral pathogens. In orderto reproduce MSD experimentally, eight pregnant sows were inoculatedintranasally with LA at day 84 of gestation. One sow gave birth to sevendead and four live but very weak piglets at day 109 of gestation; thefour live piglets died one day after birth. Another sow gave birth atday 116 to three mummified fetuses, six dead piglets and three livepiglets; two of the live piglets died within one day. A third sow gavebirth at day 117 to two mummified fetuses, eight dead and seven livepiglets. The other sows farrowed around day 115 and had less severereproductive losses. The mean number of live piglets from all eight sowsat birth was 7.3 and the mean number of dead piglets at birth was 4.6.Antibodies directed against LA were detected in 10 out of 42 serumsamples collected before the pigs had sucked. LA was isolated from threepiglets that died shortly after birth. These results justify theconclusion that LA is the causal agent of mystery swine disease.

LA grows with a cytopathic affect in pig lung macrophages and can beidentified by staining in an immuno-peroxidase-monolayer assay (IPMA)with post-infection sera of pigs c 829 and b 822, or with any of theother post-infection sera of the SPF pigs listed in table 5. Antibodiesto LA can be identified by indirect staining procedures in IPMA. LA didnot grow in any other cell system tested. LA was not neutralized byhomologous sera, or by sera directed against a set of known viruses(Table 3). LA did not haemagglutinate with the red blood cells tested.LA is smaller then 200 nm since it passes through a filter with pores ofthis size. LA is sensitive to chloroform. The above results show thatLelystad Agent is not yet identified as belonging to a certain virusgroup or other microbiological species. It has been deposited Jun. 5,1991 under number I-1102 at Institute Pasteur, France.

The genome organization, nucleotide sequences, and polypeptides derivedtherefrom, of LA have now been found. These data together with those ofothers (see below) justify classification of LA (hereafter also calledLelystad Virus or LV) as a member of a new virus family, theArteriviridae. As prototype virus of this new family we propose EquineArteritis Virus (EAV), the first member of the new family of which dataregarding the replication strategy of the genome and genome organizationbecame available (de Vries et al., 1990, and references therein). On thebasis of a comparison of our sequence data with those available forLactate Dehydrogenase-Elevating Virus (LDV; Godeny et al., 1990), wepropose that LDV is also a member of the Arteriviridae.

Given the genome organization and translation strategy of Arteriviridae,it seems appropriate to place this new virus family into the superfamilyof coronaviruses (Snijder et al., 1990a).

Arteriviruses have in common that their primary target cells inrespective hosts are macrophages. Replication of LDV has been shown tobe restricted to macrophages in its host, the mouse; whereas this strictpropensity for macrophages has not been resolved yet for EAV and LV.

Arteriviruses are spherical enveloped particles having a diameter of45-60 nm and containing an icosahedral nucleocapsid (Brinton-Darnell andPlagemann, 1975; Horzinek et al., 1971; Hyllseth, 1973).

The genome of Arteriviridae consists of a positive strandedpolyadenylated RNA molecule with a size of about 12-13 kilobases (kb)(Brinton-Darnell and Plageman, 1975; van der Zeijst et al., 1975). EAVreplicates via a 3′ nested set of six subgenomic mRNAs, ranging in sizefrom 0.8 to 3.6 kb, which are composed of a leader sequence, derivedfrom the 5′ end of the genomic RNA, which is joined to the 3′ terminalbody sequences (de Vries et al., 1990).

Here we show that the genome organization and replication strategy of LVis similar to that of EAV, coronaviruses and toroviruses, whereas thegenome sizes of the latter viruses are completely different from thoseof LV and EAV.

The genome of LV consists of a genomic RNA molecule of about 14.5 to15.5 kb in length (estimated on a neutral agarose gel), which replicatesvia a 3′ nested set of subgenomic RNAs. The subgenomic RNAs consist of aleader sequence, the length of which is yet unknown, which is derivedfrom the 5′ end of the genomic RNA and which is fused to the bodysequences derived from the 3′ end of the genomic RNA (FIG. 2).

The nucleotide sequence of the genomic RNA of LV was determined fromoverlapping cDNA clones. A consecutive sequence of 15,088 bp wasobtained covering nearly the complete genome of LV (FIG. 1, SEQ IDNO:1). In this sequence 8 open reading frames (ORFs) were identified:ORF 1A, ORF 1B, and ORFs 2 to 7.

ORF 1A and ORF 1B are predicted to encode the viral replicase orpolymerase (SEQ ID NO:2 and SEQ ID NO:3), whereas ORFs 2 to 6 arepredicted to encode structural viral membrane (envelope) associatedproteins (SEQ ID NOS:4-8). ORF 7 is predicted to encode the structuralviral nucleocapsid protein (SEQ ID NO:9).

Because the products of ORF 6 and ORF 7 of LV (SEQ ID NO:8 and SEQ IDNO:9) show a significant similarity with VpX and Vp1 of LDV,respectively, it is predicted that the sequences of ORFs 6 and 7 willalso be highly conserved among antigenic variants of LV.

The complete nucleotide sequence of FIG. 1 (SEQ ID NO:1) and all thesequences and protein products encoded by ORFs 1 to 7 (SEQ ID NOS:1-9)and possible other ORFs located in the sequence of FIG. 1 (SEQ ID NO:1)are especially suited for vaccine development, in whatever sense, andfor the development of diagnostic tools, in whatever sense. All possiblemodes are well known to persons skilled in the art.

Since it is now possible to unambiguously identify LA, the causal agentof MSD, it can now be tested whether pigs are infected with LA or not.Such diagnostic tests have, until now, been unavailable.

The test can be performed by virus isolation in macrophages, or othercell culture systems in which LA might grow, and staining the infectedcultures with antibodies directed against LA (such as post-infectionsera c 829 or b 822), but it is also feasible to develop and employother types of diagnostic tests.

For instance, it is possible to use direct or indirectimmunohistological staining techniques, i.e., with antibodies directedto LA that are labeled with fluorescent compounds such asisothiocyanate, or labeled with enzymes such as horseradish peroxidase.These techniques can be used to detect LA antigen in tissue sections orother samples from pigs suspected to have MSD. The antibodies needed forthese tests can be c 829 or b 822 or other polyclonal antibodiesdirected against LA, but monoclonal antibodies directed against LA canalso be used.

Furthermore, since the nature and organization of the genome of LA andthe nucleotide sequence of this genome have been determined, LA-specificnucleotide sequences can be identified and used to developoligonucleotide sequences that can be used as probes or primers indiagnostic techniques such as hybridization, polymerase chain reaction,or any other techniques that are developed to specifically detectnucleotide acid sequences.

It is also possible to test for antibodies directed against LA. Table 5shows that experimentally infected pigs rapidly develop antibodiesagainst LA, and table 4 shows that pigs in the field also have strongantibody responses against LA. Thus, it can now also be determinedwhether pigs have been infected with LA in the past. Such testing is ofutmost importance in determining whether pigs or pig herds or pigpopulations or pigs in whole regions or countries are free of LA. Thetest can be done by using the IPMA as described, but it is also feasibleto develop and employ other types of diagnostic tests for the detectionof antibodies directed against LA.

LA-specific proteins, polypeptides, and peptides, or peptide sequencesmimicking antigenic components of LA, can be used in such tests. Suchproteins can be derived from the LA itself, but it is also possible tomake such proteins by recombinant DNA or peptide synthesis techniques.These tests can use specific polyclonal and/or monoclonal antibodiesdirected against LA or specific components of LA, and/or use cellsystems infected with LA or cell systems expressing LA antigen. Theantibodies can be used, for example, as a means for immobilizing the LAantigen (a solid surface is coated with the antibody whereafter the LAantigen is bound by the antibody) which leads to a higher specificity ofthe test, or can be used in a competitive assay (labeled antibody andunknown antibody in the sample compete for available LA antigen).

Furthermore, the above described diagnostic possibilities can be appliedto test whether other animals, such as mammals, birds, insects or fish,or plants, or other living creatures, can be, or are, or have beeninfected with LA or related agents.

Since LA has now been identified as the causal agent of MSD, it ispossible to make a vaccine to protect pigs against this disease. Such avaccine can simply be made by growing LA in pig lung macrophagecultures, or in other cell systems in which LA grows. LA can then bepurified or not, and killed by established techniques, such asinactivation with formaline or ultra-violet light. The inactivated LAcan then be combined with adjuvantia, such as Freund's adjuvans oraluminum hydroxide or others, and this composition can then be injectedin pigs.

Dead vaccines can also be made with LA protein preparations derived fromLA infected cultures, or derived from cell systems expressingspecifically LA protein through DNA recombinant techniques. Suchsubunits of LA would then be treated as above, and this would result ina subunit vaccine.

Vaccines using even smaller components of LA, such as polypeptides,peptides, or peptides mimicking antigenic components of LA, are alsofeasible for use as dead vaccine.

Dead vaccines against MSD can also be made by recombinant DNA techniquesthrough which the genome of LA, or parts thereof, is incorporated invector systems such as vaccinia virus, herpesvirus, pseudorabies virus,adeno virus, baculo virus or other suitable vector systems that can soexpress LA antigen in appropriate cells systems. LA antigen from thesesystems can then be used to develop a vaccine as above, and pigs,vaccinated with such products would develop protective immune responsesagainst LA.

Vaccines against MSD can also be based on live preparations of LA. Sinceonly young piglets and pregnant sows seem to be seriously affected byinfection with LA, it is possible to use unattenuated LA, grown in piglung macrophages, as vaccine for older piglets, or breeding gilts. Inthis way, sows can be protected against MSD before they get pregnant,which results in protection against abortions and stillbirth, andagainst congenital infections of piglets. Also the maternal antibodythat these vaccinated sows give to their offspring would protect theiroffspring against the disease.

Attenuated vaccines (modified-live-vaccines) against MSD can be made byserially passaging LA in pig lung macrophages, in lung macrophages ofother species, or in other cell systems, or in other animals, such asrabbits, until it has lost its pathogenicity.

Live vaccines against MSD can also be made by recombinant DNA techniquesthrough which the genome of LA, or parts thereof, is incorporated invector systems such as vaccinia virus, herpesvirus, pseudorabies virus,adeno virus or other suitable vector systems that can so express LAantigen. Pigs vaccinated with such live vector systems would thendevelop protective immune responses against LA.

Lelystad Agent itself would be specifically suited to use as a livevector system. Foreign genes could be inserted in the genome of LA andcould be expressing the corresponding protein during the infection ofthe macrophages. This cell, which is an antigen-presenting cell, wouldprocess the foreign antigen and present it to B-lymphocytes andT-lymphocytes which will respond with the appropriate immune response.

Since LA seems to be very cell specific and possibly also very speciesspecific, this vector system might be a very safe system, which does notharm other cells or species.

DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:1) shows the nucleotide sequence of the LV genome. Thededuced amino acid sequence of the identified ORFs (SEQ ID NOS:2-9) areshown. The methionines encoded by the (putative) ATG start sites areindicated in bold and putative N-glycosylation sites are underlined.Differences in the nucleotide and amino acid sequence, as identified bysequencing different cDNA clones, are shown. The nucleotide sequence ofprimer 25, which has been used in hybridization experiments (see FIG. 2and section “results”), is underlined.

FIG. 2 shows the organization of the LV genome. The cDNA clones, whichhave been used for the determination of the nucleotide sequence, areindicated in the upper part of the figure. The parts of the clones,which were sequenced, are indicated in black. In the lower part of thefigure the ORFs, identified in the nucleotide sequence, and thesubgenomic set of mRNAs, encoding these ORFs are shown. The dashed linesin the ORFs represent alternative initiation sites (ATGs) of these ORFs.The leader sequence of the genomic and subgenomic RNAs is indicated by asolid box.

FIG. 3 shows the growth characteristics of LA:

-   --empty squares--titre of cell-free virus;-   --solid squares--titre of cell-associated virus;-   --solid line--percentage cytopathic effect (CPE).

MATERIALS AND METHODS

Sample Collection

Samples and pigs were collected from farms where a herd epizootic of MSDseemed to occur. Important criteria for selecting the farm as beingaffected with MSD were: sows that were off feed, the occurrence ofstillbirth and abortion, weak offspring, respiratory disease and deathamong young piglets. Samples from four groups of pigs have beeninvestigated:

-   (1) tissue samples and an oral swab from affected piglets from the    field (Table 1A);-   (2) blood samples and oral swabs from affected sows in the field    (Tables 1B and 4);-   (3) tissue samples, nasal swabs and blood samples collected from    specific-pathogen-free (SPF) pigs experimentally infected by contact    with affected sows from the field; or-   (4) tissue samples, nasal swabs and blood samples collected from    specific-pathogen-free (SPF) pigs experimentally infected by    inoculation with blood samples of affected sows from the field    (Tables 2 and 5).    Sample Preparation

Samples for virus isolation were obtained from piglets and sows which onclinical grounds were suspected to have MSD, and from experimentallyinfected SPF pigs, sows and their piglets.

Tissue samples were cut on a cryostat microtome and sections weresubmitted for direct immunofluorescence testing (IFT) with conjugatesdirected against various pig pathogens.

10% Suspensions of tissues samples were prepared in Hank's BSSsupplemented with antibiotics, and oral and nasal swabs were soaked inHank's BSS supplemented with antibiotics. After one hour at roomtemperature, the suspensions were clarified for 10 min at 6000 g and thesupernatant was stored at −70° C. for further use. Leucocyte fractionswere isolated from EDTA or heparin blood as described earlier (Wensvoortand Terpstra, 1988) and stored at −70° C. Plasma and serum for virusisolation were stored at −70° C.

Serum for serology was obtained from sows suspected to be in the acutephase of MSD, a paired serum was taken 3-9 weeks later. Furthermore,sera were taken from the experimentally infected SPF pigs at regularintervals and colostrum and serum was taken from experimentally infectedsows and their piglets. Sera for serology were stored at −20° C.

Cells

Pig lung macrophages were obtained from lungs of 5-6 weeks old SPF pigsor from lungs of adult SPF sows from the Central Veterinary Institute'sown herd. The lungs were washed five to eight times with phosphatebuffered saline (PBS). Each aliquot of washing fluid was collected andcentrifuged for 10 min at 300 g. The resulting cell pellet was washedagain in PBS and resuspended in cell culture medium (160 ml medium 199,supplemented with 20 ml 2.95% tryptose phosphate, 20 ml fetal bovineserum (FBS), and 4.5 ml 1.4% sodium bicarbonate) to a concentration of4×10⁷ cells/ml. The cell suspension was then slowly mixed with an equalvolume of DMSO mix (6.7 ml of above medium, 1.3 ml FBS, 2 mldimethylsulfoxide 97%), aliquoted in 2 ml ampoules and stored in liquidnitrogen.

Macrophages from one ampoule were prepared for cell culture by washingtwice in Earle's MEM, and resuspended in 30 ml growth medium (Earle'sMEM, supplemented with 10% FBS, 200 U/ml penicillin, 0.2 mg/mlstreptomycine, 100 U/ml mycostatin, and 0.3 mg/ml glutamine). PK-15cells (American Type Culture Collection, CCL33) and SK-6 cells (Kasza etal., 1972) were grown as described by Wensvoort et al. (1989). Secondaryporcine kidney (PK2) cells were grown in Earle's MEM, supplemented with10% FBS and the above antibiotics. All cells were grown in a cellculture cabinet at 37° C. and 5% CO².

Virus Isolation Procedures

Virus isolation was performed according to established techniques usingPK2, PK-15 and SK-6 cells, and pig lung macrophages. The former threecells were grown in 25 ml flasks (Greiner), and inoculated with the testsample when monolayers had reached 70-80% confluency. Macrophages wereseeded in 100 μl aliquots in 96-well microtiter plates (Greiner) or inlarger volumes in appropriate flasks, and inoculated with the testsample within one hour after seeding. The cultures were observed dailyfor cytopathic effects (CPE), and frozen at −70° C. when 50-70% CPE wasreached or after five to ten days of culture. Further passages were madewith freeze-thawed material of passage level 1 and 2 or higher. Somesamples were also inoculated into nine to twelve day old embryonated heneggs. Allantoic fluid was subinoculated two times using an incubationinterval of three days and the harvest of the third passage was examinedby haemagglutination at 4° C. using chicken red blood cells, and by anELISA specifically detecting nucleoprotein of influenza A viruses (DeBoer et al., 1990).

Serology

Sera were tested in haemagglutinating inhibition tests (HAI) to studythe development of antibody against haemagglutinating encephalitis virus(HEV), and swine influenza viruses H1N1 and H3N2 according to theprotocol of Masurel (1976). Starting dilutions of the sera in HAI were1:9, after which the sera were diluted twofold.

Sera were tested in established enzyme-linked immuno-sorbent assays(ELISA) for antibodies against the glycoprotein gI of pseudorabies virus(PRV; Van Oirschot et al., 1988), porcine parvo virus (PPV; Westenbrinket al., 1989), bovine viral diarrhea virus (BVDV; Westenbrink et al.,1986), and hog cholera virus (HCV; Wensvoort et al., 1988). Startingdilutions in the ELISA's were 1:5, after which the sera were dilutedtwofold.

Sera were tested for neutralizing antibodies against 30-300 TCID₅₀ ofencephalomyocarditis viruses (EMCV), porcine enteroviruses (PEV), andLelystad Agent (LA) according to the protocol of Terpstra (1978).Starting dilutions of the sera in the serum neutralization tests (SNT)were 1:5, after which the sera were diluted twofold.

Sera were tested for binding with LA in an immuno-peroxidase-monolayerassay (IPMA). Lelystad Agent (LA; code: CDI-NL-2.91) was seeded inmicrotiter plates by adding 50 ml growth medium containing 100 TCID₅₀ LAto the wells of a microtiter plate containing freshly seeded lungmacrophages. The cells were grown for two days and then fixed asdescribed (Wensvoort, 1986). The test sera were diluted 1:10 in 0.15 MNaCl, 0.05% Tween 80, 4% horse serum, or diluted further in fourfoldsteps, added to the wells and then incubated for one hour at 37° C.Sheep-anti-pig immunoglobulins (Ig) conjugated to horse radishperoxidase (HRPO, DAKO) were diluted in the same buffer and used in asecond incubation for one hour at 37° C., after which the plates werestained as described (Wensvoort et al., 1986). An intense red stainingof the cytoplasm of infected macrophages indicated binding of the serato LA.

Virus Identification Procedures

The identity of cytopathic isolates was studied by determining thebuoyant density in CsCl, by estimating particle size in negativelystained preparations through electron microscopy, by determining thesensitivity of the isolate to chloroform and by neutralizing the CPE ofthe isolate with sera with known specificity (Table 3). Whenever anisolate was specifically neutralized by a serum directed against a knownvirus, the isolate was considered to be a representative of this knownvirus.

Isolates that showed CPE on macrophage cultures were also studied bystaining in IPMA with post-infection sera of pigs c 829 or b 822. Theisolates were reinoculated on macrophage cultures and fixed at day 2after inoculation before the isolate showed CPE. Whenever an isolateshowed reactivity in IPMA with the post-infection sera of pigs c 829 orb 822, the isolate was considered to be a representative of the LelystadAgent. Representatives of the other isolates grown in macrophages oruninfected macrophages were also stained with these sera to check thespecificity of the sera.

Further Identification of Lelystad Agent

Lelystad Agent was further studied by haemagglutination at 4° C. and 37°C. with chicken, guinea pig, pig, sheep, or human O red blood cells.SIV, subtype H3N2, was used as positive control in the haemagglutinationstudies.

The binding of pig antisera specifically directed against pseudorabiesvirus (PRV), transmissible gastroenteritis virus (TGE), porcine epidemicdiarrhea virus (PED), haemagglutinating encephalitis virus (HEV),African swine fever virus (ASFV), hog cholera virus (HCV) and swineinfluenza virus (SIV) type H1N1 and H3N2, of bovine antiseraspecifically directed against bovine herpes viruses type 1 and 4 (BHV 1and 4), malignant catarrhal fever (MCF), parainfluenza virus 3 (PI3),bovine respiratory syncitial virus (BRSV) and bovine leukemia virus(BLV), and of avian antisera specifically directed against avianleukemia virus (ALV) and infectious bronchitis virus (IBV) was studiedwith species-Ig-specific HRPO conjugates in an IPMA on LA infected anduninfected pig lung macrophages as described above.

We also tested in IPMA antisera of various species directed againstmumps virus, Sendai virus, canine distemper virus, rinderpest virus,measles virus, pneumonia virus of mice, bovine respiratory syncytialvirus, rabies virus, foamy virus, maedi-visna virus, bovine and murineleukemia virus, human, feline and simian immunodeficiency virus,lymphocytic choriomeningitis virus, feline infectious peritonitis virus,mouse hepatitis virus, Breda virus, Hantaan virus, Nairobi sheep diseasevirus, Eastern, Western and Venezuelan equine encephalomyelitis virus,rubella virus, equine arteritis virus, lactic dehydrogenase virus,yellow fever virus, tick-born encephalitis virus and hepatitis C virus.

LA was blindly passaged in PK2, PK-15, and SK-6 cells, and inembryonated hen eggs. After two passages, the material was inoculatedagain into pig lung macrophage cultures for reisolation of LA.

LA was titrated in pig lung macrophages prior to and after passingthrough a 0.2 micron filter (Schleicher and Schuell). The LA wasdetected in IPMA and by its CPE. Titres were calculated according toReed and Muench (1938).

We further prepared pig antisera directed against LA. Two SPF pigs (21and 23) were infected intranasally with 10⁵ TCID₅₀ of a fifth cellculture passage of LA. Two other SPF pigs (25 and 29) were infectedintranasally with a fresh suspension of the lungs of an LA-infected SPFpiglet containing 10⁵ TCID₅₀ LA. Blood samples were taken at 0, 14, 28,and 42 days post-infection (dpi).

We further grew LA in porcine alveolar macrophages to determine itsgrowth pattern over time. Porcine alveolar macrophages were seeded inF25 flasks (Greiner), infected with LA with a multiplicity of infectionof 0.01 TCID₅₀ per cell. At 8, 16, 24, 32, 40, 48, 56, and 64 h afterinfection, one flask was examined and the percentage of CPE in relationto a noninfected control culture was determined. The culture medium wasthen harvested and replaced with an equal volume of phosphate-bufferedsaline. The medium and the flask were stored at −70° C. After allcultures had been harvested, the LA titres were determined and expressedas log TCID₅₀ ml⁻¹.

The morphology of LA was studied by electronmicroscopy. LA was culturedas above. After 48 h, the cultures were freeze-thawed and centrifugedfor 10 min at 6000×g. An amount of 30 ml supernatant was then mixed with0.3 ml LA-specific pig serum and incubated for 1.5 h at 37° C. Aftercentrifugation for 30 min at 125,000×g, the resulting pellet wassuspended in 1% Seakem agarose ME in phosphate-buffered saline at 40° C.After coagulation, the agarose block was immersed in 0.8% glutaraldehydeand 0.8% osmiumtetroxide (Hirsch et al., 1968) in veronal/acetatebuffer, pH 7.4 (230 mOsm/kg H₂O), and fixed by microwave irradiation.This procedure was repeated once with fresh fixative. The sample waswashed with water, immersed in 1% uranyl acetate, and stained bymicrowave irradiation. Throughout all steps, the sample was kept at 0°C. and the microwave (Samsung RE211D) was set at defrost for 5 min. Thinsections were prepared with standard techniques, stained with leadcitrate (Venable et al., 1965), and examined in a Philips CM 10 electronmicroscope.

We further continued isolating LA from sera of pigs originating fromcases of MSD. Serum samples originated from the Netherlands (field casethe Netherlands 2), Germany (field cases Germany 1 and Germany 2;courtesy Drs. Berner, München and Nienhoff, Münster), and the UnitedStates [experimental case United States 1 (experiment performed withATCC VR-2332; courtesy Drs. Collins, St. Paul and Chladek, St. Joseph),and field cases United States 2 and United States 3; courtesy Drs. vanAlstine, West Lafayette and Slife, Galesburg]. All samples were sent tothe “Centraal Diergeneeskundig Instituut, Lelystad” for LA diagnosis.All samples were used for virus isolation on porcine alveolarmacrophages as described. Cytophatic isolates were passaged three timesand identified as LA by specific immunostaining with anti-LA postinfection sera b 822 and c 829.

We also studied the antigenic relationships of isolates NL1 (the firstLA isolate; code CDI-NL-2.9 1), NL2, GE1, GE2, US 1, US2, and US3. Theisolates were grown in macrophages as above and were tested in IPMA witha set of field sera and two sets of experimental sera. The sera werealso tested in IPMA with uninfected macrophages.

The field sera were: Two sera positive for LV (TH-187 and TO-36) wereselected from a set of LA-positive Dutch field sera. Twenty-two serawere selected from field sera sent from abroad to Lelystad forserological diagnosis. The sera originated from Germany (BE-352, BE-392and NI-f2; courtesy Dr. Berner, München and Dr. Nienhoff, Münster), theUnited Kingdom (PA-141615, PA-141617 and PA-142440; courtesy Dr. Paton,Weybridge), Belgium (PE-1960; courtesy Prof. Pensaert, Gent), France(EA-2975 and EA-2985; courtesy Dr. Albina, Ploufragan), the UnitedStates (SL-441, SL-451, AL-RP9577, AL-P10814/33, AL-4994A, AL-7525,JC-MN41, JC-MN44 and JC-MN45; courtesy Dr. Slife, Galesburg, Dr. vanAlstine, West Lafayette, and Dr. Collins, St. Paul), and Canada (RB-16,RB-19, RB-22 and RB-23; courtesy Dr. Robinson, Quebec).

The experimental sera were: The above described set of sera of pigs 21,23, 25, and 29, taken at dpi 0, 14, 28, and 42. A set of experimentalsera (obtained by courtesy of Drs. Chladek, St. Joseph, and Collins, St.Paul) that originated from four six-month-old gilts that were challengedintranasally with 10^(5.1) TCID₅₀ of the isolate ATCC VR-2332. Bloodsamples were taken from gilt 2B at 0, 20, 36, and 63 dpi; from gilt 9Gat 0, 30, 44, and 68 dpi; from gilt 16W at 0, 25, 40, and 64 dpi; andfrom gilt 16Y at 0, 36, and 64 dpi.

To study by radio-immunoprecipitation assay (RIP; de Mazancourt et al.,1986) the proteins of LA in infected porcine alveolar macrophages, wegrew LA-infected and uninfected macrophages for 16 hours in the presenceof labeling medium containing ³⁵S-Cysteine. Then the labeled cells wereprecipitated according to standard methods with 42 dpi post-infectionsera of pig b 822 and pig 23 and with serum MN8 which was obtained 26days after infecting a sow with the isolate ATCC VR-2332 (courtesy Dr.Collins, St. Paul). The precipitated proteins were analyzed byelectrophoresis in a 12% SDS-PAGE gel and visualized by fluorography.

To characterize the genome of LA, we extracted nuclear DNA andcytoplasmatic RNA from macrophage cultures that were infected with LAand grown for 24 h or were left uninfected. The cell culture medium wasdiscarded, and the cells were washed twice with phosphate-bufferedsaline. DNA was extracted as described (Strauss, 1987). The cytoplasmicRNA was extracted as described (Favaloro et al., 1980), purified bycentrifugation through a 5.7 M CsCl cushion (Setzer et al., 1980),treated with RNase-free DNase (Pharmacia), and analyzed in a 0.8%neutral agarose gel (Moormann and Hulst, 1988).

Cloning and Sequencing

To clone LV RNA, intracellular RNA of LV-infected porcine lung alveolarmacrophages (10 μg) was incubated with 10 mM methylmercury hydroxide for10 minutes at room temperature. The denatured RNA was incubated at 42°C. with 50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 70 mM KCl, 0.5 mM dATP,dCTP, dGTP and dTTP, 0.6 μg calf thymus oligonucleotide primers pd(N)6(Pharmacia) and 300 units of Moloney murine leukemia virus reversetranscriptase (Bethesda Research Laboratories) in a total volume of 100μl 20 mM EDTA was added after 1 hr; the reaction mixture was thenextracted with phenol/chloroform, passed through a Sephadex G50 columnand precipitated with ethanol.

For synthesis of the second cDNA strand, DNA polymerase I (Boehringer)and RNase H (Pharmacia) were used (Gübler and Hoffman, 1983). Togenerate blunt ends at the termini, double-stranded cDNA was incubatedwith T4 DNA polymerase (Pharmacia) in a reaction mixture which contained0.05 mM deoxynucleotide-triphosphates. Subsequently, cDNA wasfractionated in a 0.8% neutral agarose gel (Moormann and Hulst, 1988).Fragments of 1 to 4 kb were electroeluted, ligated into the SmaI site ofpGEM-4Z (Promega), and used for transformation of Escherichia colistrain DH5α (Hanahan, 1985). Colony filters were hybridized with a³²P-labeled single-stranded cDNA probe. The probe was reversetranscribed from LV RNA which had been fractionated in a neutral agarosegel (Moormann and Hulst, 1988). Before use, the single stranded DNAprobe was incubated with cytoplasmic RNA from mock-infected lungalveolar macrophages.

The relationship between LV cDNA clones was determined by restrictionenzyme analysis and by hybridization of Southern blots of the digestedDNA with nick-translated cDNA probes (Sambrook et al., 1989).

To obtain the 3′ end of the viral genome, we constructed a second cDNAlibrary, using oligo (dT)₁₂₋₁₈ and a 3′ LV-specific oligonucleotide thatwas complementary to the minus-strand viral genome as a primer in thefirst-strand reaction. The reaction conditions for first- andsecond-strand synthesis were identical to those described above. Thislibrary was screened with virus-specific 3′ end oligonucleotide probes.

Most (>95%) of the cDNA sequences were determined with an AutomatedLaser Fluorescent A.L.F.™ DNA sequencer from Pharmacia LKB. Fluorescentoligonucleotide primer directed sequencing was performed ondouble-stranded DNA using the AutoRead™ Sequencing Kit (Pharmacia)essentially according to procedures C and D described in the Autoread™Sequencing Kit protocol. Fluorescent primers were prepared withFluorePrime™ (Pharmacia). The remaining part of the sequence wasdetermined via double-stranded DNA sequencing using oligonucleotideprimers in conjunction with a T7 polymerase based sequencing kit(Pharmacia) and α-³²S-dATP (Amersham). Sequence data were analyzed usingthe sequence analysis programs PCGENE (Intelligenetics, Inc, MountainView, USA) and FASTA (Pearson and Lipman, 1988).

Experimental Reproduction of MSD

Fourteen conventionally reared pregnant sows that were pregnant for10-11 weeks were tested for antibody against LA in the IPMA. All werenegative. Then two groups of four sows were formed and brought to theCVI. At week 12 of gestation, these sows were inoculated intranasallywith 2 ml LA (passage level 3, titre 10^(4.8) TCID₅₀/ml). Serum and EDTAblood samples were taken at day 10 after inoculation. Food intake,rectal temperature, and other clinical symptoms were observed daily. Atfarrowing, the date of birth and the number of dead and living pigletsper sow were recorded, and samples were taken for virus isolation andserology.

Results

Immunofluorescence

Tissue sections of pigs with MSD were stained in an IFT withFITC-conjugates directed against African swine fever virus, hog choleravirus, pseudorabies virus, porcine parvo virus, porcine influenza virus,encephalomyocarditis virus and Chlamydia psittaci. The sections werestained, examined by fluorescent microscopy and all were found negative.

Virus Isolation from Piglets from MSD Affected Farms

Cytopathic isolates were detected in macrophage cultures inoculated withtissue samples of MSD affected, two-to-ten day old piglets. Sixteen outof 19 piglets originating from five different farms were positive (Table1A). These isolates all reacted in IPMA with the post-infection serum ofpig c 829, whereas non-inoculated control cultures did not react. Theisolates, therefore, were representatives of LA. One time a cytopathicisolate was detected in an SK-6 cell culture inoculated with asuspension of an oral swab from a piglet from a sixth farm (farm VE)(Table 1A). This isolate showed characteristics of the picorna viridaeand was neutralized by serum specific for PEV 2, therefore, the isolatewas identified as PEV 2 (Table 3). PK2, PK-15 cells and hen eggsinoculated with samples from this group remained negative throughout.

Virus Isolation from Sows from MSD Affected Farms

Cytopathic isolates were detected in macrophage cultures inoculated withsamples of MSD affected sows. 41 out of 63 sows originating from 11farms were positive (Table 1B). These isolates all reacted in IPMA withthe post-infection serum of pig b 822 and were, therefore,representatives of LA. On one occasion a cytopathic isolate was detectedin a PK2 cell culture inoculated with a suspension of a leucocytefraction of a sow from farm HU (Table 1B). This isolate showedcharacteristics of the picorna viridae and was neutralized by serumspecific for EMCV, therefore, the isolate was identified as EMCV (Table3). SK-6, PK-15 cells and hen eggs inoculated with samples from thisgroup remained negative.

Virus Isolation from SPF Pigs Kept in Contact with MSD Affected Sows

Cytopathic isolates were detected in macrophage cultures inoculated withsamples of SPF pigs kept in contact with MSD affected sows. Four of the12 pigs were positive (Table 2). These isolates all reacted in IPMA withthe post-infection serum of pig c 829 and of pig b 822 and were,therefore, representatives of LA. Cytopathic isolates were also detectedin PK2, PK-15 and SK-6 cell cultures inoculated with samples of theseSPF pigs. Seven of the 12 pigs were positive (Table 2), these isolateswere all neutralized by serum directed against PEV 7. One of these sevenisolates was studied further and other characteristics also identifiedthe isolate as PEV 7 (Table 3).

Virus Isolation from SPF Pigs Inoculated with Blood of MSD Affected Sows

Cytopathic isolates were detected in macrophage cultures inoculated withsamples of SPF pigs inoculated with blood of MSD affected sows. Two outof the eight pigs were positive (Table 2). These isolates all reacted inIPMA with the post-infection serum of pig c 829 and of pig b 822 andwere, therefore, representatives of LA. PK2, SK-6 and PK-15 cellsinoculated with samples from this group remained negative.

Summarizing, four groups of pigs were tested for the presence of agentsthat could be associated with mystery swine disease (MSD).

In group one, MSD affected piglets, the Lelystad Agent (LA) was isolatedfrom 16 out of 20 piglets; one time PEV 2 was isolated.

In group two, MSD affected sows, the Lelystad Agent was isolated from 41out of 63 sows; one time EMCV was isolated. Furthermore, 123 out of 165MSD affected sows seroconverted to the Lelystad Agent, as tested in theIPMA. Such massive seroconversion was not demonstrated against any ofthe other viral pathogens tested.

In group three, SPF pigs kept in contact with MSD affected sows, LA wasisolated from four of the 12 pigs; PEV 7 was isolated from seven pigs.All 12 pigs seroconverted to LA and PEV7.

In group four, SPF pigs inoculated with blood of MSD affected sows, theLA was isolated from two pigs. All eight pigs seroconverted to LA.

Serology of Sows from MSD Affected Farms

Paired sera from sows affected with MSD were tested against a variety ofviral pathogens and against the isolates obtained during this study(Table 4). An overwhelming antibody response directed against LA wasmeasured in the IPMA (75% of the sows seroconverted, in 23 out of the 26farms seroconversion was found), whereas with none of the other viralpathogens a clear pattern of seroconversion was found. Neutralizingantibody directed against LA was not detected.

Serology of SPF Pigs Kept in Contact with MSD Affected Sows

All eight SPF pigs showed an antibody response in the IPMA against LA(Table 5). None of these sera were positive in the IPMA performed onuninfected macrophages. None of these sera were positive in the SNT forLA. The sera taken two weeks after contact had all high neutralizingantibody titres (>1280) against PEV 7, whereas the pre-infection serawere negative (<10), indicating that all pigs had also been infectedwith PEV 7.

Serology of SPF Pigs Inoculated with Blood of MSD Affected Sows

All eight SPF pigs showed an antibody response in the IPMA against LA(Table 5). None of these sera were positive in the IPMA performed onuninfected macrophages. None of these sera were positive in the SNT forLA. The pre- and two weeks post-inoculation sera were negative (<10)against PEV 7.

Further Identification of Lelystad Agent

LA did not haemagglutinate with chicken, guinea pig, pig, sheep, orhuman O red blood cells.

LA did not react in IPMA with sera directed against PRV, TGE, PED, ASFV,etc.

After two blind passages, LA did not grow in PK2, PK-15, or SK-6 cells,or in embryonated hen eggs, inoculated through the allantoic route.

LA was still infectious after it was filtered through a 0.2 micronfilter, titres before and after filtration were 10^(5.05) and 10^(5.3)TCID₅₀ as detected by IPMA.

Growth curve of LA (see FIG. 3). Maximum titres of cell-free virus wereapproximately 10^(5.5) TCID₅₀ ml⁻¹ from 32-48 h after inoculation. Afterthat time the macrophages were killed by the cytopathic effect of LA.

Electronmicroscopy. Clusters of spherical LA particles were found. Theparticles measured 45-55 nm in diameter and contained a 30-35 nmnucleocapsid that was surrounded by a lipid bilayer membrane. LAparticles were not found in infected cultures that were treated withnegative serum or in negative control preparations.

Isolates from the Netherlands, Germany, and the United States. All sevenisolates were isolated in porcine alveolar macrophages and passagedthree to five times. All isolates caused a cytopathic effect inmacrophages and could be specifically immunostained with anti-LA sera b822 and the 42 dpi serum 23. The isolates were named NL2, GE1, GE2, US1,US2, and US3.

Antigenic relationships of isolates NL1, NL2, GE1, GE2, US1, US2, andUS3. None of the field sera reacted in IPMA with uninfected macrophagesbut all sera contained antibodies directed against one or more of theseven isolates (Table 7). None of the experimental sera reacted in IPMAwith uninfected macrophages, and none of the 0 dpi experimental serareacted with any of the seven isolates in IPMA (Table 8). All seven LAisolates reacted with all or most of the sera from the set ofexperimental sera of pigs 21, 23, 25, and 29, taken after 0 dpi. Onlythe isolates US1, US2, and US3 reacted with all or most of the sera fromthe set of experimental sera of gilts 2B, 9G, 16W, and 16Y, taken after0 dpi.

Radioimmunoprecipitation studies. Seven LA-specific proteins weredetected in LA-infected macrophages but not in uninfected macrophagesprecipitated with the 42 dpi sera of pigs b 822 and 23. The proteins hadestimated molecular weights of 65, 39, 35, 26, 19, 16, and 15kilodalton. Only two of these LA-specific proteins, of 16 and 15kilodalton, were also precipitated by the 26 dpi serum MN8.

Sequence and Organization of the Genome of LV

The nature of the genome of LV was determined by analyzing DNA and RNAfrom infected porcine lung alveolar macrophages. No LV-specific DNA wasdetected. However, we did detect LV-specific RNA. In a 0.8% neutralagarose gel, LV RNA migrated slightly slower than a preparation of hogcholera virus RNA of 12.3 kb (Moormann et al., 1990) did. Although noaccurate size determination can be performed in neutral agarose gels, itwas estimated that the LV-specific RNA is about 14.5 to 15.5 kb inlength.

To determine the complexity of the LV-specific RNAs in infected cellsand to establish the nucleotide sequence of the genome of LV, weprepared cDNA from RNA of LV-infected porcine lung alveolar macrophagesand selected and mapped LV-specific cDNA clones as described underMaterials and Methods. The specificity of the cDNA clones wasreconfirmed by hybridizing specific clones, located throughout theoverlapping cDNA sequence, to Northern blots carrying RNA of LV-infectedand uninfected macrophages. Remarkably, some of the cDNA cloneshybridized with the 14.5 to 15.5 kb RNA detected in infected macrophagesonly, whereas others hybridized with the 14.5 to 15.5 kb RNA as well aswith a panel of 4 or 5 RNAs of lower molecular weight (estimated size, 1to 4 kb). The latter clones were all clustered at one end of the cDNAmap and covered about 4 kb of DNA. These data suggested that the genomeorganization of LV may be similar to that of coronaviridae (Spaan etal., 1988), Berne virus (BEV; Snijder et al., 1990b), a torovirus, andEAV (de Vries et al., 1990), i.e., besides a genomic RNA there aresubgenomic mRNAs which form a nested set which is located at the 3′ endof the genome. This assumption was confirmed when sequences of the cDNAclones became available and specific primers could be selected to probethe blots with. A compilation of the hybridization data obtained withcDNA clones and specific primers, which were hybridized to Northernblots carrying the RNA of LV-infected and uninfected macrophages, isshown in FIG. 2. Clones 12 and 20 which are located in the 5′ part andthe centre of the sequence, respectively, hybridize to the 14.5 to 15.5kb genomic RNA detected in LV-infected cells only. Clones 41 and 39,however, recognize the 14.5 to 15.5 kb genomic RNA and a set of 4 and 5RNAs of lower molecular weight, respectively. The most instructive andconclusive hybridization pattern, however, was obtained with primer 25,which is located at the ultimate 5′ end in the LV sequence (compare FIG.1). Primer 25 hybridized to a panel of 7 RNAs, with an estimatedmolecular weight ranging in size from 0.7 to 3.3 kb (subgenomic mRNAs),as well as the genomic RNA. The most likely explanation for thehybridization pattern of primer 25 is that 5′ end genomic sequences, thelength of which is yet unknown, fuse with the body of the mRNAs whichare transcribed from the 3′ end of the genome. In fact, thehybridization pattern obtained with primer 25 suggests that 5′ endgenomic sequences function as a so called “leader sequence” insubgenomic mRNAs. Such a transcription pattern is a hallmark ofreplication of coronaviridae (Spaan et al., 1988), and of EAV (de Vrieset al., 1990).

The only remarkable discrepancy between LV and EAV which could beextracted from the above data is that the genome size of LV is about 2.5kb larger than that of EAV.

The consensus nucleotide sequence of overlapping cDNA clones is shown inFIG. 1 (SEQ ID NO:1). The length of the sequence is 15,088 basepairs,which is in good agreement with the estimated size of the genomic LVRNA.

Since the LV cDNA library was made by random priming of the reversetranscriptase reaction with calf thymus pd(N) 6 primers, no cDNA cloneswere obtained which started with a poly-A stretch at their 3′ end. Toclone the 3′ end of the viral genome, we constructed a second cDNAlibrary, using oligo (dT) and primer 39U183R in the reversetranscriptase reaction. Primer 39U183R is complementary to LVminus-strand RNA, which is likely present in a preparation of RNAisolated from LV-infected cells. This library was screened withvirus-specific probes (nick-translated cDNA clone 119 andoligonucleotide 119R64R), resulting in the isolation of five additionalcDNA clones (e.g., cDNA clone 151, FIG. 2). Sequencing of these cDNAclones revealed that LV contains a 3′ poly(A) tail. The length of thepoly(A) tail varied between the various cDNA clones, but its maximumlength was twenty nucleotides. Besides clone 25 and 155 (FIG. 2), fouradditional cDNA clones were isolated at the 5′ end of the genome, whichwere only two to three nucleotides shorter than the ultimate 5′nucleotide shown in FIG. 1 (SEQ ID NO:1). Given this finding and giventhe way cDNA was synthesized, we assume to be very close to the 5′ endof the sequence of LV genomic RNA.

Nearly 75% of the genomic sequence of LV encodes ORF 1A and ORF 1B. ORF1A probably initiates at the first AUG (nucleotide position 212, FIG. 1)encountered in the LV sequence. The C-terminus of ORF 1A overlaps theputative N-terminus of ORF 1B over a small distance of 16 nucleotides.It thus seems that translation of ORF 1B proceeds via ribosomalframeshifting, a hallmark of the mode of translation of the polymeraseor replicase gene of coronaviruses (Boursnell et al., 1987; Bredenbeeket al. 1990) and the torovirus BEV (Snijder et al., 1990a). Thecharacteristic RNA pseudoknot structure which is predicted to be formedat the site of the ribosomal frameshifting is also found at thislocation in the sequence of LV (results not shown).

ORF 1B encodes an amino acid sequence (SEQ ID NO:3) of nearly 1400residues which is much smaller than ORF 1B of the coronaviruses MHV andIBV (about 3,700 amino acid residues; Bredenbeek et al., 1990; Boursnellet al., 1987) and BEV (about 2,300 amino acid residues; Snijder et al.,1990a). Characteristic features of the ORF 1B product (SEQ ID NO:3) ofmembers of the superfamily of coronaviridae, like the replicase motifand the Zinc finger domain, can also be found in ORF 1B of LV (resultsnot shown).

Whereas ORF 1A and ORF 1B encode the viral polymerase (SEQ ID NO:2 andSEQ ID NO:3) and, therefore, are considered to encode a non-structuralviral protein, ORFs 2 to 7 are believed to encode structural viralproteins (SEQ ID NOS:4-9).

The products of ORFs 2 to 6 (SEQ ID NOS:4-8) all show featuresreminiscent of membrane (envelope) associated proteins. ORF 2 encodes aprotein (SEQ ID NO:4) of 249 amino acids containing two predictedN-linked glycosylation sites (Table 9). At the N-terminus a hydrophobicsequence, which may function as a so-called signal sequence, isidentified. The C-terminus also ends with a hydrophobic sequence, whichin this case may function as a transmembrane region, which anchors theORF 2 product (SEQ ID NO:4) in the viral envelope membrane.

ORF 3 may initiate at the AUG starting at nucleotide position 12394 orat the AUG starting at nucleotide position 12556 and then encodesproteins (SEQ ID NO:5) of 265 and 211 amino acids, respectively. Theprotein of 265 residues contains seven putative N-linked glycosylationsites, whereas the protein of 211 residues contains four (Table 9). Atthe N-terminus of the protein (SEQ ID NO:5) of 265 residues ahydrophobic sequence is identified.

Judged by hydrophobicity analysis, the topology of the protein encodedby ORF 4 (SEQ ID NO:6) is similar to that encoded by ORF 2 (SEQ ID NO:4)if the product of ORF 4 (SEQ ID NO:6) initiates at the AUG starting atnucleotide position 12936. However, ORF 4 may also initiate at two otherAUG codons (compare FIGS. 1 and 2) starting at positions 12981 and 13068in the sequence respectively. Up to now it is unclear which start codonis used. Depending on the start codon used, ORF 4 may encode proteins(SEQ ID NO:6) of 183 amino acids containing four putative N-linkedglycosylation sites, of 168 amino acids containing four putativeN-linked glycosylation sites, or of 139 amino acids containing threeputative N-linked glycosylation sites (Table 9).

ORF 5 is predicted to encode a protein (SEQ ID NO:7) of 201 amino acidshaving two putative N-linked glycosylation sites (Table 9). Acharacteristic feature of the ORF 5 product (SEQ ID NO:7) is theinternal hydrophobic sequence between amino acid 108 to amino acid 132.

Analysis for membrane spanning segments and hydrophilicity of theproduct of ORF 6 (SEQ ID NO:8) shows that it contains threetransmembrane spanning segments in the N-terminal 90 amino acids of itssequence. This remarkable feature is also a characteristic of the smallenvelope glycoprotein M or E1 of several coronaviruses, e.g., InfectiousBronchitis Virus (IBV; Boursnell et al., 1984) and Mouse Hepatitis Virus(MHV: Rottier et al., 1986). It is, therefore, predicted that theprotein encoded by ORF 6 (SEQ ID NO:8) was a membrane topology analogousto that of the M or E1 protein of coronaviruses (Rottier et al., 1986).A second characteristic of the M or E1 protein is a so-called surfacehelix which is located immediately adjacent to the presumed thirdtransmembrane region. This sequence of about 25 amino acids which isvery well conserved among coronaviruses is also recognized, althoughmuch more degenerate, in LV. Yet we predict the product of LV ORF 6 (SEQID NO:8) to have an analogous membrane associated function as thecoronavirus M or E1 protein. Furthermore, the protein encoded by ORF 6(SEQ ID NO:8) showed a strong similarity (53% identical amino acids)with VpX (Godeny et al., 1990) of LDV.

The protein encoded by ORF 7 (SEQ ID NO:9) has a length of 128 aminoacid residues (Table 9) which is 13 amino acids longer than Vp1 of LDV(Godeny et al., 1990). Yet a significant similarity (43% identical aminoacids) was observed between the protein encoded by ORF 7 (SEQ ID NO:9)and Vp1. Another shared characteristic between the product of ORF 7 (SEQID NO:9) and Vp1 is the high concentration of basic residues (Arg, Lysand His) in the N-terminal half of the protein. Up to amino acid 55, theLV sequence contains 26% Arg, Lys and His. This finding is fully in linewith the proposed function of the ORF 7 product (SEQ ID NO:9) or Vp1(Godeny et al., 1990), namely encapsidation of the viral genomic RNA. Onthe basis of the above data, we propose the LV ORF 7 product (SEQ IDNO:9) to be the nucleocapsid protein N of the virus.

A schematic representation of the organization of the LV genome is shownin FIG. 2. The map of overlapping clones used to determine the sequenceof LV is shown in the top panel. A linear compilation of this mapindicating the 5′ and 3′ end of the nucleotide sequence of LV, shown inFIG. 1 (SEQ ID NO:1), including a division in kilobases, is shown belowthe map of cDNA clones and allows the positioning of these clones in thesequence. The position of the ORFs identified in the LV genome isindicated below the linear map of the LV sequence. The bottom panelshows the nested set of subgenomic mRNAs, and the position of these RNAsrelative to the LV sequence.

In line with the translation strategy of coronavirus, torovirus andarterivirus subgenomic mRNAs, it is predicted that ORFs 1 to 6 aretranslated from the unique 5′ end of their genomic or mRNAs. This uniquepart of the mRNAs is considered to be that part of the RNA that isobtained when a lower molecular weight RNA is “subtracted” from thehigher molecular weight RNA which is next in line. Although RNA 7 formsthe 3′ end of all the other genomic and subgenomic RNAs, and thus doesnot have a unique region, it is believed that ORF 7 is only translatedfrom this smallest sized mRNA. The “leader sequence” at the 5′ end ofthe subgenomic RNAs is indicated with a solid box. The length of thissequence is about 200 bases, but the precise site of fusion with thebody of the genomic RNAs still has to be determined.

Experimental Reproduction of MSD

Eight pregnant sows were inoculated with LA and clinical signs of MSDsuch as inappetance and reproductive losses were reproduced in thesesows. From day four to day 10-12 post-inoculation (p.i.), all sowsshowed a reluctance to eat. None of the sows had elevated bodytemperatures. Two sows had bluish ears at day 9 and 10 p.i. In Table 6the day of birth and the number of living and dead piglets per sow isgiven. LA was isolated from 13 of the born piglets.

TABLE 1 Description and results of virus isolation of field samples. ASamples of piglets suspected of infection with MSD. number age farm ofpigs days material used results* RB 5 2 lung, tonsil, and brains  5 × LADV 4 3 lung, brains, pools of kidney,  3 × LA spleen TH 3 3-5 lung,pools of kidney, tonsil  3 × LA DO 3 10  lung, tonsil  2 × LA ZA 4 1lung, tonsil  3 × LA VE 1 ? oral swab  1 × PEV 2 TOTAL 20 16 × LA,  1 ×PEV 2 B Samples of sows suspected of infection with MSD. number farm ofsows material used results TH 2 plasma and leucocytes  1 × LA HU 5plasma and leucocytes  2 × LA, 1 × EMCV TS 10 plasma and leucocytes  6 ×LA HK 5 plasma and leucocytes  2 × LA LA 6 plasma and leucocytes  2 × LAVL 6 serum and leucocytes  5 × LA TA 15 serum 11 × LA LO 4 plasma andleucocytes  2 × LA JA 8 plasma and leucocytes  8 × LA VD 1 plasma andleucocytes  1 × LA VW 1 serum  1 × LA TOTAL 63 41 × LA, 1 × EMCV*Results are given as the number of pigs from which the isolation wasmade. Sometimes the isolate was detected in more than one sample perpig. LA = Lelystad Agent PEV 2 = porcine entero virus type 2 EMCV =encephalomyocarditis virus

TABLE 2 Description and results of virus isolation of samples of pigswith experimentally induced infections. sow pig@ material used results*A (LO) # c 835 lung, tonsil 2 × LA c 836 nasal swabs 2 × PEV 7 c 837nasal swabs B (JA) c 825 lung, tonsil c 821 nasal swabs 1 × PEV 7 c 823nasal swabs 4 × PEV 7 C (JA) c 833 lung, tonsil 1 × LA, 1 × PEV 7 c 832nasal swabs 2 × PEV 7 c 829 nasal swabs, plasma 3 × LA, 2 × PEV 7 andleucocytes D (VD) c 816 lung, tonsil c 813 nasal swabs 1 × LA c 815nasal swabs 1 × PEV 7 TOTAL isolates from contact pigs 7 × LA, 13 × PEV7 A b 809 nasal swabs b 817 nasal swabs B b 818 nasal swabs, plasma 1 ×LA and leucocytes b 820 nasal swabs C b 822 nasal swabs b 826 nasalswabs D b 830 nasal swabs 1 × LA b 834 nasal swabs TOTAL isolates fromblood 2 × LA inoculated pigs @SPF pigs were either kept in contact (c)with a sow suspected to be infected with MSD, or were given 10 ml EDTAblood (b) of that sow intramuscularly at day 0 of the experiment. Groupsof one sow and three SPF pigs (c) were kept in one pen, and all four ofthese groups were housed in one stable. At day 6, one SPF pig in eachgroup was killed and tonsil and lungs were used for virus isolation. Thefour groups of SPF pigs inoculated with blood (b) were housed in fourother pens in a separate stable. Nasal swabs of the SPF pigs were takenat day 2, 5, 7 and 9 of the experiment, and EDTA blood for virusisolation from plasma and leucocytes was taken whenever a pig had fever.*Results are given as number of isolates per pig. LA = Lelystad AgentPEV 7 = porcine entero virus type 7 # In brackets the initials of thefarm of origin of the sow are given.

TABLE 3 Identification of viral isolates buoyant¹ density sens³ toneutralized by⁴ origin and size in particle² chloro- serum directed cellculture in CsCl FM (nm) form against (titre) leucocytes 1.33 g/ml 28-30not EMCV (1280) sow farm HU sens. PK-15, PK2, SK6 oral swab 28-30 notPEV 2 (>1280) piglet farm VEND sens. SK6 nasal swabs, tonsil ND 28-30not PEV 7 (>1280) SPF pigs CVI sens. PK-15, PK2, SK6 various samples1.19 g/ml pleomorf sens. none (all <5) various farms pig lungmacrophages ¹Buoyant density in preformed linear gradients of CsCl inPBS was determined according to standard techniques (Brakke; 1967).Given is the density where the peak of infectivity was found. ²Infectedand noninfected cell cultures of the isolate under study werefreeze-thawed. Cell lysates were centrifuged for 30 min at 130,000 g,the resulting pellet was negatively stained according to standardtechniques (Brenner and Horne; 1959), and studied with a Philips CM 10electron microscope. Given is the size of particles that were present ininfected and not present in non-infected cultures. ³Sensitivity tochloroform was determined according to standard techniques (Grist, Ross,and Bell; 1974). ⁴Hundred to 300 TCID⁵⁰ of isolates were mixed withvarying dilutions of specific antisera and grown in the appropriate cellsystem until full CPE was observed. Sera with titres higher than 5 wereretested, and sera which blocked with high titres the CPE wereconsidered specific for the isolate. The isolates not sensitive tochloroform were tested with sera specifically directed against porcineentero viruses (PEV) 1 to 11 (courtesy Dr. Knowles, Pirbright, UK),against encephalomyocarditis virus (EMCV; courtesy Dr. Ahl, Tübingen,Germany), against porcine parvo virus, and against swine vesiculardisease. The isolate (code: CDI-NL-2.91) sensitive to chloroform wastested with antisera specifically directed against pseudorabies virus,bovine herpes virus 1, bovine herpes virus 4, malignant catarrhal virus,bovine viral diarrhea virus, hog cholera virus, swine influenza virusH1N1 and H3N2, parainfluenza 3 virus, bovine respiratory syncitialvirus, transmissible gastroenteritis virus, porcine epidemic diarrhoeavirus, haemagglutinating encephalitis virus, infectious bronchitisvirus, bovine leukemia virus, avian leukemia virus, maedi-visna virus,and with the experimental sera obtained from the SPF-pigs (see Table 5).

TABLE 4 Results of serology of paired field sera taken from sowssuspected to have MSD. Sera were taken in the acute phase of the diseaseand 3-9 weeks later. Given is the number of sows which showed a fourfoldor higher rise in titre/number of sows tested. Interval^(i) HAI ELISAFarm in weeks HEV H1N1 H3N2 PPV PPV BVDV HCV TH 3 0/6 0/6 0/6 0/6 0/60/5 0/6 RB 5  0/13  1/13  0/13 1/9 0/7 0/6 0/9 HU 4 0/5 0/5 3/5 0/5 0/50/5 0/5 TS 3  1/10  0/10  0/10  0/10  0/10 0/4  0/10 VL 3 0/5 0/5 0/50/5 1/5 0/5 0/5 JA 3  0/11  1/11  3/11  0/11  2/11  0/11  0/11 WE 4 1/61/6 1/6 3/7 3/7 0/7 0/7 GI 4 0/4 1/4 0/4 0/4 0/4 0/4 0/4 SE 5 0/8 0/80/8 0/8 0/6 0/3 0/8 KA 5 0/1 0/1 0/1 0/1 0/1 ND 0/1 HO 3 1/6 0/5 1/6 0/60/6 0/6 0/6 NY 4 0/5 1/5 1/5 0/3 0/4 0/2 0/4 JN 3  0/10  5/10  0/10 0/10  1/10  0/10  0/10 KO^(f) 3  1/10  0/10  0/10  0/10  2/10  0/10 0/10 OE 9 ND ND ND 0/6 0/6 0/6 0/6 LO 6 ND ND ND 0/3 0/3 0/2 0/3 WI 4ND ND ND 0/1 1/1 0/1 0/3 RR 3 ND ND ND 1/8 0/8 0/8 0/8 RY 4 ND ND ND 0/30/4 0/3 0/4 BE 5 ND ND ND  0/10  0/10  0/10  0/10 BU 3 ND ND ND 1/6 0/60/6 0/6 KR 3 ND ND ND 1/4 0/4 0/4 0/4 KW 5 ND ND ND  0/10  0/10  0/10 0/10 VR 5 ND ND ND 1/6 0/6 0/6 0/6 HU 4 ND ND ND 1/4 0/3 0/3 0/4 ME 3ND ND ND 0/5 1/5 0/5 0/5 total negative^(n) 19 41 29 97  16 140 165total positive^(p) 77 48 62 55 131  1  0 total sero-converted^(s)  4 10 9  9  11  0  0 total tested 100  99 100  161  158 141 165 The sera weretested in haemagglutinating inhibition (HAI) tests for the detection ofantibody against haemagglutinating encephalitis virus (HEV), and swineinfluenza viruses H1N1 and H3N2, in enzyme-linked-immuno sorbent assays(ELISA) for the detection of antibody against the glycoprotein gI ofpseudorabies virus (PRV), against porcine parvo virus (PPV), bovineviral diarrhea virus (BVDV), and hog cholera virus (HCV). Interval SNTIPMA Farm in weeks EMCV EMCVi PEV2 PEV2i PEV7 PEV7i LA LA TH 3 0/6 0/60/5 0/5 0/6 0/5 0/6 6/6 RB 5 1/7 1/9 0/6 2/6 1/8 0/6  0/13 7/9 HU 4 ND0/5 0/5 0/5 ND 0/5 0/5 5/5 TS 3  0/10  0/10 0/7 0/4  0/10 0/7 ND 10/10VL 3 ND ND 1/5 0/5 ND 0/5 ND 5/5 JA 3  0/11  0/11  0/11  0/11  1/11 2/11 0/5  8/11 WE 4 1/7 1/6 1/6 1/7 1/7 1/7 0/7 7/7 GI 4 0/4 0/4 0/40/4 0/4 0/4 0/4 4/4 SE 5 0/8 0/8 0/6 1/8 0/8 1/5 0/8 6/8 KA 5 0/1 0/10/1 0/1 0/1 0/1 0/1 0/1 HO 3 0/6 0/6 0/6 0/6 0/6 0/6 0/6 4/6 NY 4 0/40/4 0/2 0/2 0/4 0/3 0/4 4/4 JN 3  0/10  0/10  1/10 0/9  0/10  0/10  0/10 5/10 KO^(f) 3  0/10  0/10  2/10  2/10  1/10  3/10 ND  8/10 OE 9 0/6 0/61/6 1/5 ND 1/6 ND 4/6 LO 6 0/3 0/3 0/3 0/3 0/3 0/3 ND 3/3 WI 4 ND ND 0/10/1 ND 0/1 ND 0/3 RR 3 0/8 1/8 0/8 0/8 0/8 0/8 ND 8/8 RY 4 0/4 ND 0/40/1 ND 1/4 ND 1/4 BE 5 ND ND  0/10  0/10 ND  1/10 ND  0/10 BU 3 ND ND0/6 0/6 ND 0/6 ND 6/6 KR 3 ND ND 0/4 0/4 ND 0/4 ND 1/4 KW 5 ND ND  0/10 0/10 ND  1/10 ND 10/10 VR 5 ND ND 0/6 1/6 ND 0/6 ND 6/6 HU 4 ND ND 0/30/4 ND 0/3 ND 3/4 ME 3 ND ND 0/5 0/5 ND 0/5 ND 2/5 total neg.^(n) 15 29 0  0  2  1 69  15 total pos.^(p) 88 74 144 138 90 136  0  27 totalsero-  2  3  6  8  4  10  0 123 converted^(s) total tested 105  107  150146 96 147 69 165 The sera were tested in serum neutralization tests(SNT) for the detection of neutralizing antibody directed againstencephalomyocarditis virus (EMCV), the isolated (i) EMCV, porcine enteroviruses (PEV) 2 and 7 and the PEV isolates (i), and against the LelystadAgent (LA), and were tested in an immuno-peroxidase-monolayer-assay(IPMA) for the detection of antibody directed against the Lelystad Agent(LA). ^(f)fattening pigs. ^(i)time between sampling of the first andsecond serum. ^(n)total number of pigs of which the first serum wasnegative in the test under study, and of which the second serum was alsonegative or showed a less than fourfold rise in titre. ^(p)total numberof pigs of which the first serum was positive and of which the secondserum showed a less than fourfold rise in titre. ^(s)total number ofpigs of which the second serum had a fourfold or higher titre than thefirst serum in the test under study. ND = not done.

TABLE 5 Development of antibody directed against Lelystad Agent asmeasured by IPMA. A contact pigs serum titres in IPMA Weeks postcontact: Pig 0 2 3 4 5 c 836 0 10 640 640 640 c 837 0 10 640 640 640 c821 0 640 640 640 640 c 823 0 160 2560 640 640 c 829 0 160 640 1024010240 c 832 0 160 640 640 2560 c 813 0 640 2560 2560 2560 c 815 0 160640 640 640 B blood inoculated pigs serum titres in IPMA Weeks postinoculation: Pig 0 2 3 4 6 b 809 0 640 2560 2560 2560 b 817 0 160 640640 640 b 818 0 160 640 640 640 b 820 0 160 640 640 640 b 822 0 640 25602560 10240 b 826 0 640 640 640 10240 b 830 0 640 640 640 2560 b 834 0160 640 2560 640 See Table 2 for description of the experiment. All pigswere bled at regular intervals and all sera were tested in animmuno-peroxidase-monolayer-assay (IPMA) for the detection of antibodydirected against the Lelystad Agent (LA).

TABLE 6 Experimental reproduction of MSD. No. of piglets at birth alivedead LA¹ in piglets Length of (Number No. of born died in Sow gestationAb pos)² deaths week 1 dead week 1 52 113 12 (5) 3 (2) 6 2 4 965 116 3(0) 9 (3) 2 4 997 114 9 (0) 1 (0) 0 1305 116 7 (0) 2 (0) 1 134 109 4 (4)7 (4) 4 3 941 117 7 10 1056 113 7 (1) 3 (0) 4 1065 115 9 2 ¹LA wasisolated from lung, liver, spleen, kidney, or ascitic fluids.²Antibodies directed against LA were detected in serum samples takenbefore the piglets had sucked, or were detected in ascitic fluids ofpiglets born dead.

TABLE 7 Reactivity in IPMA of a collection of field sera from Europe andNorth America tested with LA isolates from the Netherlands (NL1 andNL2), Germany (GE1 and GE2), and the United States (US1, US2 and US3).Isolates: NL1 NL2 GE1 GE2 US1 US2 US3 Sera from: The Netherlands TH-1873.5_(t) 3.5 2.5 3.5 — — — TO-36 3.5 3.0 2.5 3.0 — 1.0 — Germany BE-3524.0 3.5 2.5 3.0 — 1.5 — BE-392 3.5 3.5 2.5 2.5 1.5 1.5 0.5 NI-f2 2.5 1.52.0 2.5 — — — United Kingdom PA-141615 4.0 3.0 3.0 3.5 — — — PA-1416174.0 3.5 3.0 3.5 — 2.5 2.0 PA-142440 3.5 3.0 2.5 3.5 — 2.0 2.5 BelgiumPE-1960 4.5 4.5 3.0 4.0 1.5 — — France EA-2975 4.0 3.5 3.0 3.0 2.0 — —EA-2985 3.5 3.0 3.0 2.5 — — — United States SL-441 3.5 1.5 2.5 2.5 3.53.5 3.0 SL-451 3.0 2.0 2.5 2.5 3.5 4.5 4.0 AL-RP9577 1.5 — — 1.0 3.0 4.02.5 AL-P10814/330.5 2.5 — — 2.5 3.5 3.0 AL-4094A — — — — 1.0 2.0 0.5AL-7525 — — — — — 1.0 — JC-MN41 — — — — 1.0 3.5 1.0 JC-MN44 — — — — 2.03.5 2.0 JC-MN45 — — — — 2.0 3.5 2.5 Canada RB-16 2.5 — 3.0 2.0 3.0 3.5 —RB-19 1.0 — 1.0 — 2.5 1.5 — RB-22 1.5 — 2.0 2.5 2.5 3.5 — RB-23 — — — —— 3.0 — _(t)= titre expressed as negative log; — = negative

TABLE 8 Reactivity in IPMA of a collection of experimental sera raisedagainst LA and SIRSV tested with LA isolates from the Netherlands (NL1and NL2), Germany (GE1 and GE2), and the United States (US1, US2 andUS3). Isolates: NL1 NL2 GE1 GE2 US1 US2 US3 Sera: anti-LA: 21 14 dpi2.5^(t) 2.0 2.5 3.0 1.5 2.0 1.5 28 dpi 4.0 3.5 3.5 4.0 — 2.5 1.5 42 dpi4.0 3.5 3.0 3.5 1.5 2.5 2.0 23 14 dpi 3.0 2.0 2.5 3.0 1.0 2.0 1.0 28 dpi3.5 3.5 3.5 4.0 1.5 2.0 2.0 42 dpi 4.0 4.0 3.0 4.0 — 2.5 2.5 25 14 dpi2.5 2.0 2.5 3.0 1.5 2.0 1.0 28 dpi 4.0 3.5 4.0 3.5 — 1.5 2.0 42 dpi 3.54.0 3.5 3.5 1.5 2.0 2.0 29 14 dpi 3.5 3.5 3.0 3.5 — 2.0 1.5 28 dpi 3.53.5 3.0 3.5 — 2.5 2.0 42 dpi 4.0 3.5 3.5 4.0 1.5 2.5 2.5 anti-SIRSV: 2B20 dpi — — — — 2.0 2.0 — 36 dpi — — — — 1.5 2.0 — 63 dpi — — — — 1.0 1.0— 9G 30 dpi — — — — 2.5 3.0 — 44 dpi — — — — 2.5 3.5 — 68 dpi — — — —2.0 3.5 1.5 16W 25 dpi — — — — 2.0 3.0 — 40 dpi — — — — 2.0 3.0 — 64 dpi— — — — 2.5 2.5 1.5 16Y 36 dpi — — — — 1.0 3.0 1.0 64 dpi — — — — 2.53.0 — ^(t)= titer expressed as negative log; — = negative

TABLE 9 Characteristics of the ORFs of Lelystad Virus. Calculated No. ofsize of the number of Nucleotides amino unmodified glycosylation ORF(first-last) acids peptide (kDa) sites ORF1A  212-7399 2396 260.0 3 (SEQID NO: 2) ORF1B  7384-11772 1463 161.8 3 (SEQ ID NO: 3) ORF2 11786-12532249 28.4 2 (SEQ ID NO: 4) ORF3 12394-13188 265 30.6 7 (SEQ ID NO: 5)12556-13188 211 24.5 4 ORF4 12936-13484 183 20.0 4 (SEQ ID NO: 6)12981-13484 168 18.4 4 13068-13484 139 15.4 3 ORF5 13484-14086 201 22.42 (SEQ ID NO: 7) ORF6 14077-14595 173 18.9 2 (SEQ ID NO: 8) ORF714588-14971 128 13.8 1 (SEQ ID NO: 9)

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Table Showing % Identity of Selected PRRSV Strains with vr 2332 at theNucleotide Level

vr 2332 vr 2428 vr 2429 vr 2430 vr 2431 vr 2475 nucleotides isu 12 isu22 isu 55 isu 3927 isu 1894 ORF 1a  191-7702 unavailable unavailableunavailable unavailable unavailable ORF 1b  7699-12072 unavailableunavailable unavailable unavailable unavailable ORF 2 12074-12844 96 9897 94 97 ORF 3 12697-13461 93 98 93 89 97 ORF 4 13242-13778 93 98 94 9197 ORF 5 13789-14391 92 98 91 92 97 ORF 6 14376-14900 98 98 97 94 99 ORF7 14890-15264 98 99 97 95 98 vr 2332 vr 2428 vr 2429 vr 2430 vr 2431 vr2475 sequences from sequences from sequences from sequences fromsequences from sequences from medline U.S. Pat. No. medline medlinemedline medline accession 6592873 FIG. accession accession accessionaccession number 20 (ORFs 2-4) numbers numbers numbers numbers AY150564and SEQ ID 13 U34297 (ORFs U34299 (ORFs U34298 (ORFs U34296 (ORFs (ORFs5-7) 2-5) and 2-5) and 2-5) and 2-5) and U18749 (ORFs U18751 (ORFsU18750 (ORFs U18748 (ORFs 6 & 7) 6 & 7) 6 & 7) 6 & 7)Data Showing % Identity of Selected PRRSV Strains with vr 2332 at theNucleotide Level

VR 2332 ORF Compared with BLAST RID NO: No. ISU Nos.1089390113-19392-160251220478.BLASTQ4 2 22, 55, 3297, 18941089393263-9055-107144512008.BLASTQ4 2 121089391007-14686-210393370361.BLASTQ4 3 22, 55, 3297, 18941089393390-12324-67292085899.BLASTQ4 3 121089391107-17943-59202408632.BLASTQ4 4 22, 55, 3297, 18941089394015-28604-101796745891.BLASTQ4 4 121089391477-27885-45601462313.BLASTQ4 5 22, 55, 3297, 18941089391915-9312-36320692317.BLASTQ4 5 121089391570-1608-200472981478.BLASTQ4 6 22, 55, 3297, 18941089392011-9288-167646260344.BLASTQ4 6 121089391744-5002-113924068691.BLASTQ4 7 22, 55, 3297, 18941089393088-4599-31943670528.BLASTQ4 7 12

1. A method of producing an immunogenic composition, the methodcomprising: introducing a purified preparation containing apolynucleotide encoding at least one polypeptide encoded by one or moreopen reading frames (ORFs) of ORFs 1-7 of a mystery swine disease virus,wherein the mystery swine disease virus is the isolate Lelystad Agent(CDI-NL-2.91) deposited Jun. 5, 1991 with the Institut Pasteur,Collection Nationale de Cultures De Microorganismes (C.N.C.M.) 25, ruedu Docteur Roux, 75724--Paris Cedex 15, France, deposit number I-1102,into a suitable host cell; culturing said suitable host cell; andisolating one of the following therefrom: a suitable host cellcontaining said polypeptide; virus; viral protein; viral polynucleicacid; or mixtures thereof.
 2. The method according to claim 1, furthercomprising isolating at least one of a cultured host cell and apolypeptide encoded by said polynucleotide.
 3. The method according toclaim 1, wherein introducing a purified preparation containing apolynucleotide encoding at least one polypeptide encoded by one or moreopen reading frames (ORFs) of ORFs 1-7 of a mystery swine disease virus,into a suitable host cell comprises infecting the suitable host cellwith a virus comprising the polynucleotide.
 4. The method according toclaim 1, wherein the mystery swine disease virus is characterized asbeing specifically reactive with serum antibodies of a sow, said serumantibodies obtained by intranasally inoculating a specific pathogen freesow with two milliliters of the virus identified as deposit number11102, deposited Jun. 5, 1991 with the Insitut Pasteur, Paris, France(at passage level 3, titer 10.sup.4.8 TCID.sub.50/milliliter) andcollecting serum antibodies from the thus inoculated sow after 25 to 33days.
 5. The method according to claim 4, further comprising isolatingat least one of a cultured host cell and a polypeptide encoded by saidpolynucleotide.